1. Field of Invention
The present invention relates to a method for detecting and/or for quantifying poly(A) RNA and/or mRNA, wherein a poly(dT) oligonucleotide which comprises a fluorescent dye and also a quencher is hybridized to the nucleic acid to be detected. Non-hybridized poly(dT) oligonucleotide is degraded by an added nuclease, and as a result, fluorescently labelled nucleotides are released and the resulting fluorescent signal is measured.
2. Description of Related Art
The quantification of the exact amount of mRNA added is of great importance for many methods in molecular biology. More particularly for various gene expression analyses, such as, for example, quantitiative reverse-transcription PCR (qRT-PCR) and microarrays, this so-called normalization is important for obtaining accurate and comparable experimental results.
The amount of RNA added is usually determined by means of spectrophotometry by measuring the absorbance at 260 nm. This technique, however, has the disadvantage that, at this wavelength, not only the mRNA but also further nucleic acids, such as DNA, rRNA, tRNA and further non-coding RNAs, are detected, i.e. the exact amount of mRNA present is not known.
For this reason, the DNA is generally removed from the sample by means of a purification method prior to the quantification of the RNA, meaning additional process steps and costs. After the removal of the DNA, the amount of total RNA is determined in a spectrophotometer and, from this, the amount of mRNA present is indirectly deduced. Since, however, the ratio of mRNA to total RNA is not constant, but can be completely different from preparation to preparation and also in different starting materials, a high error rate arises when the total RNA is used to deduce the amount of the mRNA present.
A further disadvantage of this method is that it cannot be established whether the mRNA in the sample has already (partially) been degraded. The degradation of mRNAs often begins with the removal of the poly(A) tail at the 3′ end and continues with the degradation of the coding region. Such mRNA is no longer intact and often no longer usable for molecular biology experiments.
In order to be able to add intact, quantifiable total mRNA to a molecular biology reaction, this mRNA first has to be purified from the biological sample in a time-consuming and multi-step procedure with the aid of poly(dT) nucleotides coupled to a solid phase. This procedure is, however, not quantitative, and so, during the purification procedure, some of the mRNA from the biological sample is lost. After the purification, the mRNA thus isolated has to be quantified spectrophotometrically, meaning not only an additional process step but also a loss of some of the isolated mRNA.